1 The human P2Y 11 (hP2Y 11 ) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y 11 ) and Chinese hamster ovary cells (CHO-hP2Y 11 ), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2 In 1321N1-hP2Y 11 cells, ATP promoted inositol phosphate (IP) accumulation with low mM potency (EC 50 =8.5+0.1 mM), whereas it was 15 fold less potent (130+10 mM) in evoking cyclic AMP production. 3 In CHO-hP2Y 11 cells, ATP promoted IP accumulation with slightly higher potency (EC 50 =3.6+1.3 mM) than in 1321N1-hP2Y 11 cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC 50 =62.4+15.6 mM) than for IP accumulation. Comparable di erences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4 In 1321N1-hP2Y 11 and CHO-hP2Y 11 cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60 ± 80% (P50.001) with no change in its potency. Likewise, chelation of intracellular Ca 2+ decreased ATP-promoted cyclic AMP accumulation by *45% in 1321N1-hP2Y 11 cells, whereas chelation had no e ect on either the e cacy or potency of ATP in CHO-hP2Y 11 cells. 5 We conclude that coupling of hP2Y 11 receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca 2+ mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y 11 and CHO-hP2Y 11 cells. British Journal of Pharmacology (2001) 132, 318 ± 326 Keywords: ATP; cyclic AMP; inositol phosphates; P2Y receptors; P2Y 11 receptor; protein kinase C; Ca 2+ mobilization Abbreviations: 1321N1-hD1, 1321N1 cells expressing the human D1 dopamine receptor; 1321N1-hP2Y 11 , 1321N1 cells expressing the human P2Y 11 receptor; ATPgS, adenosine 5'-triphosphate-g-thiophosphate; BAPTA-AM, 1,2-bis(oaminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; CHO-hP2Y 11 , CHO-K1 cells expressing the human P2Y 11 receptor; CHO-K1 cells, Chinese hamster ovary cells; DMEM, Dulbecco's Modi®ed Eagle's Medium; hD1 receptor, human D1 dopamine receptor; hP2Y 11 receptor, human P2Y 11
The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars, and cysteinyl leukotrienes, and coupled to intracellular Ca 21 mobilization and inhibition of cAMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the hemagglutinintagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1, or Chinese hamster ovary (CHO) cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cAMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (.80%) of forskolinstimulated cAMP accumulation in C6 and CHO cells expressing the P2Y 14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or human embryonic kidney 293 cells transiently transfected with GPR17 alone or cotransfected with Ga q/i5 , which links G icoupled receptors to the G q -regulated phospholipase C (PLC) signaling pathway, or PLC«, which is activated by the Ga 12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, coexpression experiments of GPR17 with cysteinyl leukotriene receptor 1 (CysLTR1) suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose, and LTC4 are not the cognate ligands of GPR17.
Eight human G protein-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), P2Y(13), and P2Y(14)) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I(sc)) or Ca(2+) mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y(1), P2Y(11), P2Y(12), and P2Y(14) receptors reside at the basolateral membrane, P2Y(2), P2Y(4), and P2Y(6) receptors are expressed at the apical membrane, and the P2Y(13) receptor is unsorted. Biotinylation studies and I(sc) measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G(q)-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11)) in lung and colonic epithelial cells (16HBE14o- and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells.
UTP is a potent full agonist at both the human P2Y 4 (hP2Y 4 ) and rat P2Y 4 (rP2Y 4 ) receptor. In contrast, ATP is a potent full agonist at the rP2Y 4 receptor but is a similarly potent competitive antagonist at the hP2Y 4 receptor. To delineate the structural determinants of agonism versus antagonism in these species homologues, we expressed a series of human/rat P2Y 4 receptor chimeras in 1321N1 human astrocytoma cells and assessed the capacity of ATP and UTP to mobilize intracellular Ca 2؉ . Replacement of the NH 2 terminus of the hP2Y 4 receptor with the corresponding region of the rP2Y 4 receptor resulted in a receptor that was activated weakly by ATP, whereas replacement of the second extracellular loop (EL2) of the hP2Y 4 receptor with that of the rP2Y 4 receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP, albeit with lower potencies than those observed at the rP2Y 4 receptor. These potencies were increased, and ATP was converted to a full agonist by replacing both the NH 2 terminus and EL2 in the hP2Y 4 receptor with the corresponding regions from the rP2Y 4 receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids, Asn-177, Ile-183, and Leu-190, contribute to the capacity of EL2 to impart ATP agonism. Taken together, these results suggest that the second extracellular loop and the NH 2 terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y 4 receptors. Extracellular nucleotides elicit diverse physiological effects by activating G protein-coupled P2Y receptors (1, 2). Molecular cloning and heterologous receptor expression studies have led to the identification and characterization of eight human P2Y (hP2Y) 1 receptor subtypes (hP2Y 1,2,4,6,11-14 ). hP2Y 1 , hP2Y 2 , hP2Y 4 , hP2Y 6 , and hP2Y 11 receptors display 27-52% amino acid identity and couple via heterotrimeric G proteins of the G q family to the activation of phospholipase C, generation of inositol phosphates, and mobilization of intracellular Ca 2ϩ stores (2-4). In addition to coupling to phospholipase C, the hP2Y 11 receptor also couples to G s to activate adenylyl cyclase and promotes cyclic AMP accumulation (5-7). The recently identified P2Y 12 , P2Y 13 , and P2Y 14 receptors, which are encoded on a short segment of chromosome 3, have high sequence identity with each other (40 -48%) but share relatively little sequence identity (22-25%) with the other hP2Y receptors. The P2Y 12 receptor has been shown to be the G i -coupled receptor in platelets that, together with the P2Y 1 receptor, mediate ADP-promoted platelet aggregation (8 -11). P2Y 13 and P2Y 14 receptors are also coupled to G i and are activated by ADP and UDPglucose, respectively (12, 13).Differences in nucleotide selectivity have been observed between species orthologues of P2Y receptors. For example, the avian p2y3 and rat P2Y 6 receptor are species homologues with ϳ65% identity that diffe...
P2Y 2 and P2Y 4 receptors, which have 52% sequence identity, are both expressed at the apical membrane of Madin-Darby canine kidney cells, but the locations of their apical targeting signals are distinctly different. The targeting signal of the P2Y 2 receptor is located between the N terminus and 7TM, whereas that of the P2Y 4 receptor is present in its C-terminal tail. To identify the apical targeting signal in the P2Y 2 receptor, regions of the P2Y 2 receptor were progressively substituted with the corresponding regions of the P2Y 4 receptor lacking its targeting signal. Characterization of these chimeras and subsequent mutational analysis revealed that four amino acids (Arg 95 , Gly 96 , Asp 97 , and Leu 108 ) in the first extracellular loop play a major role in apical targeting of the P2Y 2 receptor. Mutation of RGD to RGE had no effect on P2Y 2 receptor targeting, indicating that receptor-integrin interactions are not involved in apical targeting. P2Y 2 receptor mutants were localized in a similar manner in Caco-2 colon epithelial cells. This is the first identification of an extracellular protein-based targeting signal in a seven-transmembrane receptor.Extracellular nucleotides play an important role in a broad range of physiological and pathophysiological processes through two classes of receptors, the ligand-gated P2X receptors and the G protein-coupled P2Y receptors (1, 2). To date, pharmacological characterization and molecular cloning have identified seven P2X receptors (P2X 1-7 ) and eight P2Y receptor subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ). The P2Y receptors can be divided into two main subfamilies based on sequence identity and phylogenetic considerations: the P2Y 1 receptor subfamily, which is comprised of P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, and the P2Y 12 receptor subfamily, comprising P2Y 12 , P2Y 13 , and P2Y 14 receptors. The P2Y 1 receptor subfamily is coupled to activation of phospholipase C, generation of inositol phosphates, activation of protein kinase C, and mobilization of intracellular Ca 2ϩ stores. In addition to coupling to activation of phospholipase C, the P2Y 11 receptor is also coupled to G s , thereby activating adenylyl cyclase (3-5). In contrast, the three members of the P2Y 12 receptor subfamily couple to G i/o and therefore inhibit adenylyl cyclase (6 -8).Although P2Y receptors mediate a multitude of cellular responses throughout the body, one of their main functions is to regulate ion transport in polarized epithelial cells. Proteins in polarized epithelial cells can be expressed predominantly at either the apical or basolateral membrane surface, or they can be indiscriminatingly distributed to both membranes. The proper targeting of proteins to their respective membrane surface is critical in host defense, nutrient absorption, ion transport, and signal transduction. However, the mechanism(s) by which epithelial cells target proteins to a particular membrane surface remains a critical question in epithelial cell b...
SummaryThe P2Y 1 receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y 1 receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or dihydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.
The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y15 receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca2+ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9[12]). However, the cell line (HEK293) used to carry out those studies endogenously expresses A2A and A2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93 [15]) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, α-ketoglutarate. Consistent with this report, α-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts α-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by α-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.
Diadenosine 5,5ٟ-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC 50 values ranging between 5 and 15 M. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y 11 , exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC 50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.The dinucleoside diphosphates diadenosine 5Ј,5ٞ-P1,P2-diphosphate (Ap2A), 2 adenosine guanosine diphosphate (Ap2G), and diguanosine diphosphate (Gp2G) represent a new class of growth-promoting extracellular mediators present in platelet secretory granules (1) and in cardiac myocytes (2), capable of stimulating cardiac myocyte proliferation (1) and believed to play a role in the control of cardiovascular tone (3). The intraplatelet concentration of each one of these dinucleotides has been estimated to be in the range between 30 and 100 M (1). It has also been shown that the concentration of Ap2A, Ap2G, and Gp2G in the supernatant of platelets stimulated with 0.05 units/ml thrombin is ϳ60% of the total intraplatelet amount of each dinucleoside diphosphate, suggesting that their primary function is extracellular (1). The enzyme responsible for their synthesis has not been identified, and the effect of these dinucleotides on platelet function has not yet been investigated.We have recently demonstrated that ADP-ribosyl cyclases from Axinella polypoides, (Porifera, Demospongiae), Aplysia californica (Molluscs), and human CD38 can synthesize three adenylic dinucleotides from cyclic ADP-ribose and adenine (Ade): Ap2A and two isomers thereof, called P18 and P24, which are characterized by an unusual N-glycosidic bond between one adenine a...
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